Method of preparing a food product from cruciferous seeds

ABSTRACT

A method of preparing a food product rich in glucosinolates wherein cruciferous seeds, with the exception of cabbage, cress, mustard and radish seeds, are germinated, and sprouts are harvested prior to the 2-leaf stage, to form a food product containing a plurality of sprouts.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grant PO1 CA44530, entitled "Novel Strategies for Chemoprotection Against Cancer",(Paul Talalay, Principal Investigator) awarded by the National CancerInstitute, Department of Health and Human Services.

BACKGROUND OF THE INVENTION

I. Field of Invention

This invention relates to a dietary approach to reducing the level ofcarcinogens in animals and their cells and thereby reducing the risk ofdeveloping cancer. In particular, this invention relates to theproduction and consumption of foods which are rich in cancerchemoprotective compounds. More specifically, this invention relates tochemoprotective compounds that modulate mammalian enzymes which areinvolved in metabolism of carcinogens. This invention relates to foodsources which are extremely rich in compounds that induce the activityof Phase 2 enzymes, without inducing biologically significant activitiesof those Phase 1 enzymes that activate carcinogens.

II. Background

It is widely recognized that diet plays a large role in controlling therisk of developing cancers and that increased consumption of fruits andvegetables reduces cancer incidence in humans. It is believed that amajor mechanism of protection depends on the presence of chemicalcomponents in plants that, when delivered to mammalian cells, elevatelevels of Phase 2 enzymes that detoxify carcinogens.

Early studies on the mechanism of chemoprotection by certain chemicalsassumed that these chemoprotectors induced activities of monooxygenases,also known as Phase 1 enzymes or cytochromes P-450. However, Talalay etal., reviewed in "Chemical Protection Against Cancer by Induction ofElectrophile Detoxication (Phase II) Enzymes" In: CELLULAR AND MOLECULARTARGETS OF CHEMOPREVENTION, L. Wattenberg et al., CRC Press, Boca Raton,Fla., pp 469-478 (1992)! determined that administration of the knownchemoprotector butylated hydoxyanisole (BHA) to rodents resulted inlittle change in cytochromes P-450 (Phase 1 enzyme) activities, butprofoundly elevated Phase 2 enzymes. Phase 2 enzymes such as glutathionetransferases, NAD(P)H:quinone reductase (QR) andglucuronosyltransferases, detoxify DNA-damaging electrophilic forms ofultimate carcinogens. Selective inducers of Phase 2 enzymes aredesignated monofunctional inducers. Prochaska & Talalay, Cancer Res. 48:4776-4782 (1988). The monofunctional inducers are nearly allelectrophiles and belong to 8 distinct chemical classes including (1)diphenols, phenylenediamines and quinones; (2) Michael reactionacceptors containing olefins or acetylenes conjugated toelectron-withdrawing groups; (3) isothiocyanates; (4)1,2-dithiole-3-thiones; (5) hydroperoxides; (6) trivalent inorganic andorganic arsenic derivatives; (7) heavy metals with potencies related totheir affinities for thiol groups including Hg²⁺, and Cd²⁺ ; and (8)vicinal dimercaptans. Prestera et al., Proc. Natl. Acad. Sci. USA 90:2963-2969 (1993). The only apparent common property shared by all ofthese inducers is their ability to react with thiol groups.

Chemoprotective agents can be used to reduce the susceptibility ofmammals to the toxic and neoplastic effects of carcinogens. Thesechemoprotectors can be of plant origin or synthetic compounds. Syntheticanalogs of naturally occurring inducers have also been generated andshown to block chemical carcinogenesis in animals. Posner et al., J.Med. Chem. 37: 170-176 (1994); Zhang et al., Proc. Natl. Acad. Sci. USA91: 3147-3150 (1994); Zhang et al., Cancer Res. (Suppl) 54: 1976s-1981s(1994).

Highly efficient methods have been developed for measuring the potencyof plant extracts to increase or induce the activities of Phase 2enzymes. Prochaska & Santamaria, Anal. Biochem. 169: 328-336 (1988) andProchaska et al., Proc. Natl. Acad. Sci. USA 89: 2394-2398 (1992). Inaddition, these methods have been employed for isolating the compoundsresponsible for the inducer activities in plants and for evaluating theanticarcinogenic activities of these compounds and their syntheticanalogs. Zhang et al., Proc. Natl. Acad. Sci. USA 89: 2399-2403 (1992)and Posner et al., J. Med. Chem. 17: 170-176 (1994).

Although inducer activity has been found in many different families ofedible plants, the amounts are highly variable, depending on family,genus, species, variety, or cultivar of the plant selection and ongrowth and harvesting conditions. Thus, there is a need in the art toidentify particular edible plants and methods of growing and preparingthem that yield high levels of Phase 2 enzyme-inducer activity forchemoprotection. There is also a need for methods of growing andpreparing edible plants that produce a known spectrum of specificinducers of Phase 2 enzyme activity in order to increase the efficiencywith which specific carcinogens, or classes of carcinogens, are targetedfor inactivation. In addition, there is a need for methods of plantbreeding and selection to increase the level of Phase 2 inducer activityand to manipulate the spectrum of inducers produced in particularcultivars.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide food products andfood additives that are rich in cancer chemoprotective compounds.

Another object of the present invention is to provide food productswhich contain substantial quantities of Phase 2 enzyme-inducers and areessentially free of Phase 1 enzyme-inducers.

It is a further object of the present invention to provide food productswhich contain substantial quantities of Phase 2 enzyme-inducingpotential and non-toxic levels of indole glucosinolates and theirbreakdown products and goitrogenic hydroxybutenyl glucosinolates.

These objects, and others, are achieved by providing cruciferoussprouts, with the exception of cabbage, cress, mustard and radishsprouts, harvested prior to the 2-leaf stage. The cruciferous sproutsinclude Brassica oleracea varieties acephala, alboglabra, botrytis,costata, gemmifera, gongylodes, italica, medullosa, palmifolia, ramosa,sabauda, sabellica, and selensia.

Another embodiment of the present invention provides cruciferoussprouts, with the exception of cabbage, cress, mustard and radishsprouts, harvested prior to the 2-leaf stage, wherein the sprouts aresubstantially free of Phase 1 enzyme-inducing potential.

Yet another embodiment of the present invention provides a non-toxicsolvent extract of cruciferous sprouts, with the exception of cabbage,cress, mustard and radish sprouts, harvested prior to the 2-leaf stage.The non-toxic solvent extract can be a water extract. In addition, thewater extract can comprise a cruciferous vegetable, such as acruciferous vegetable of the genus Raphanus, comprising an activemyrosinase enzyme.

Another embodiment of the present invention provides a food productcomprising cruciferous sprouts, with the exception of cabbage, cress,mustard and radish sprouts, harvested prior to the 2-leaf stage;extracts of the sprouts or cruciferous seeds; or any combination of thesprouts or extracts.

A further embodiment of the present invention provides a method ofincreasing the chemoprotective amount of Phase 2 enzymes in a mammal,comprising the step of administering an effective quantity ofcruciferous sprouts, with the exception of cabbage, cress, mustard andradish sprouts, harvested prior to the 2-leaf stage.

Yet another embodiment of the present invention provides a method ofincreasing the chemoprotective amount of Phase 2 enzymes in a mammal,comprising the step of administering an effective quantity of a foodproduct comprising cruciferous sprouts, with the exception of cabbage,cress, mustard and radish sprouts, harvested prior to the 2-leaf stage.

Another embodiment of the present invention provides cruciferous sproutsharvested prior to the 2-leaf stage, wherein the sprouts have at least200,000 units per gram fresh weight of Phase 2 enzyme-inducing potentialwhen measured after 3 days of growth from seeds that produce saidsprouts and contain non-toxic levels of indole glucosinolates and theirbreakdown products and goitrogenic hydroxybutenyl glucosinolates. Thecruciferous sprouts include Brassica oleracea varieties acephala,alboglabra, botrytis, costata, gemmifera, gongylodes, italica,medullosa, palmifolia, ramosa, sabauda, sabellica, and selensia.

A further embodiment of the present invention provides a food productcomprising sprouts harvested prior to the 2-leaf stage, wherein thesprouts have at least 200,000 units per gram fresh weight of Phase 2enzyme-inducing potential when measured after 3 days from growth ofseeds that produce the sprouts and contain non-toxic levels of indoleglucosinolates and their breakdown products and goitrogenichydroxybutenyl glucosinolates; extracts of the sprouts or cruciferousseeds; or any combination of the sprouts or extracts.

Yet another embodiment of the present invention provides cruciferoussprouts harvested prior to the 2-leaf stage, wherein the sprouts have atleast 200,000 units per gram fresh weight of Phase 2 enzyme-inducingpotential when measured after 3 days of growth from seeds that producethe sprouts and contain non-toxic levels of indole glucosinolates andtheir breakdown products and goitrogenic hydroxybutenyl glucosinolatesand are substantially free of Phase 1 enzyme-inducing potential.

Another embodiment of the present invention provides a non-toxic solventextract of cruciferous sprouts harvested prior to the 2-leaf stage,wherein the sprouts have at least 200,000 units per gram fresh weight ofPhase 2 enzyme-inducing potential when measured after 3 days of growthfrom seeds that produce the sprouts and contain non-toxic levels ofindole glucosinolates and their breakdown products and goitrogenichydroxybutenyl glucosinolates. The non-toxic solvent extract can be awater extract. In addition, the water extract can comprise a cruciferousvegetable, such as a cruciferous vegetable of the genus Raphanus,comprising an active myrosinase enzyme.

Yet another embodiment of the present invention provides a method ofincreasing the chemoprotective amount of Phase 2 enzymes in a mammal,comprising the step of administering an effective quantity ofcruciferous sprouts harvested prior to the 2-leaf stage, wherein thesprouts have at least 200,000 units per gram fresh weight of Phase 2enzyme-inducing potential when measured after 3 days of growth fromseeds that produce the sprouts and contain non-toxic levels of indoleglucosinolates and their breakdown products and goitrogenichydroxybutenyl glucosinolates.

Yet another embodiment of the present invention provides a method ofincreasing the chemoprotective amount of Phase 2 enzymes in a mammal,comprising the step of administering an effective quantity of a foodproduct comprising sprouts harvested prior to the 2-leaf stage, whereinthe sprouts have at least 200,000 units per gram fresh weight of Phase 2enzyme-inducing potential when measured after 3 days of growth fromseeds that produce the sprouts and contain non-toxic levels of indoleglucosinolates and their breakdown products and goitrogenichydroxybutenyl glucosinolates.

A further embodiment of the present invention provides a method ofpreparing a food product rich in glucosinolates, comprising germinatingcruciferous seeds, with the exception of cabbage, cress, mustard andradish seeds, and harvesting sprouts prior to the 2-leaf stage to form afood product comprising a plurality of sprouts. The cruciferous sproutsinclude Brassica oleracea varieties acephala, alboglabra, botrytis,costata, gemmifera, gongylodes, italica, medullosa, palmifolia, ramosa,sabauda, sabellica, and selensia and contain non-toxic levels of indoleglucosinolates and their breakdown products and goitrogenichydroxybutenyl glucosinolates.

Yet another embodiment of the present invention provides a food productrich in glucosinolates made by germinating cruciferous seeds, with theexception of cabbage, cress, mustard and radish seeds, and harvestingsprouts prior to the 2-leaf stage to form a food product comprising aplurality of sprouts.

Yet another embodiment of the present invention provides a method ofpreparing a food product comprising extracting glucosinolates andisothiocyanates from cruciferous sprouts, with the exception of cabbage,cress, mustard and radish sprouts, harvested prior to the 2-leaf stage,with a non-toxic solvent and recovering the extracted glucosinolates andisothiocyanates. Myrosinase enzyme, or a vegetable, such as Raphanusspecies, containing the enzyme is mixed with the cruciferous sprouts,the extract, or both the sprouts and the extract.

An embodiment of the present invention provides a method of preparing afood product rich in glucosinolates, comprising germinating cruciferousseeds having at least 200,000 units per gram fresh weight of Phase 2enzyme-inducing potential when measured after 3 days of growth fromseeds that produce the sprouts and which contain non-toxic levels ofindole glucosinolates and their breakdown products and goitrogenichydroxybutenyl glucosinolates, and harvesting sprouts prior to the2-leaf stage to form a food product comprising a plurality of sprouts.The seeds may be Brassica oleracea, including the varieties acephala,alboglabra, botrytis, costata, gemmifera, gongylodes, italica,medullosa, palmifolia, ramosa, sabauda, sabellica, and selensia.

Yet another embodiment of the present invention provides a food productrich in glucosinolates made by germinating cruciferous seeds having atleast 200,000 units per gram fresh weight of Phase 2 enzyme-inducingpotential when measured after 3 days of growth from seeds that producethe sprouts and which contain non-toxic levels of indole glucosinolatesand their breakdown products and goitrogenic hydroxybutenylglucosinolates, and either harvesting sprouts at the 2-leaf stage toform a food product comprising a plurality of sprouts. The nutritionalproduct contains non-toxic levels of indole glucosinolates and theirbreakdown products and goitrogenic hydroxybutenyl glucosinolates.

A further embodiment of the present invention provides a method ofpreparing a food product comprising extracting glucosinolates andisothiocyanates with a solvent from cruciferous seeds, sprouts, plantsor plant parts, wherein seeds that produce the sprouts, plants or plantparts producing sprouts having at least 200,000 units per gram freshweight of Phase 2 enzyme-inducing potential when measured after 3 daysof growth and wherein the seeds, sprouts, plants or plant parts havenon-toxic levels of indole glucosinolates and their breakdown productsand goitrogenic hydroxybutenyl glucosinolates, and recovering theextracted glucosinolates and isothiocyanates. The non-toxic extractionsolvent can be water. Myrosinase enzyme, or a vegetable, such asRaphanus species, containing the enzyme is mixed with the cruciferoussprouts, seeds, plants, plant parts or extract, or any combinationthereof.

A further embodiment of the present invention provides a method ofreducing the level of carcinogens in mammals, comprising administeringcruciferous sprouts, with the exception of cabbage, cress, mustard andradish sprouts.

Yet another embodiment of the present invention provides a method ofreducing the level of carcinogens in mammals, comprising administeringcruciferous sprouts having at least 200,000 units per gram fresh weightof Phase 2 enzyme-inducing potential when measured after 3 days ofgrowth from seeds that produce the sprouts and non-toxic levels ofindole glucosinolates and their breakdown products and goitrogenichydroxybutenyl glucosinolates.

Another embodiment of the present invention provides a method ofpreparing a food product by introducing cruciferous seeds, having atleast 200,000 units per gram fresh weight of Phase 2 enzyme-inducingpotential when measured after 3 days of growth from seeds that producethe sprouts and non-toxic levels of indole glucosinolates andgoitrogenic hydroxybutenyl glucosinolates, into an edible ingredient.

A further embodiment of the present invention provides a method ofextracting glucosinolates and isothiocyanates from plant tissue whichcomprises homogenizing the plant tissue in an excess of a mixture ofdimethyl sulfoxide, acetonitrile, and dimethylformamide (DMF/ACN/DMSO)at a temperature that prevents myrosinase activity.

Another embodiment of the present invention provides cruciferous sproutsharvested prior to the 2-leaf stage, wherein the ratio of monofunctionalto bifunctional inducers is at least 20 to 1.

Another object of the present invention is to provide a food productsupplemented with a purified or partially purified glucosinolate.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the total inducing potential of organic solvent extracts ofbroccoli and daikon cultivars as a function of age.

FIG. 2 shows the high resolution NMR spectra of isolated glucosinolatesobtained from hot aqueous extracts of 3-day old Saga broccoli sprouts.

DETAILED DESCRIPTION

I. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

A bifunctional inducer is a molecule which increases activities of bothPhase 1 enzymes such as cytochromes P-450 and Phase 2 enzymes andrequires the participation of Aryl hydrocarbon (Ah) receptor and itscognate Xenobiotic Response Element (XRE). Examples include flat planararomatics such as polycyclic hydrocarbons, azo dyes or2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD).

A chemoprotector or chemoprotectant is a synthetic or naturallyoccurring chemical agent that reduces susceptibility in a mammal to thetoxic and neoplastic effects of carcinogens.

A food product is any ingestible preparation containing the sprouts ofthe instant invention, or extracts or preparations made from thesesprouts, which are capable of delivering Phase 2 inducers to the mammalingesting the food product. The food product can be freshly preparedsuch as salads, drinks or sandwiches containing sprouts of the instantinvention. Alternatively, the food product containing sprouts of theinstant invention can be dried, cooked, boiled, lyophilized or baked.Breads, teas, soups, cereals, pills and tablets, are among the vastnumber of different food products contemplated.

Inducer activity or Phase 2 enzyme-inducing activity is a measure of theability of a compound(s) to induce Phase 2 enzyme activity. In thepresent invention, inducer activity is measured by means of the murinehepatoma cell bioassay of QR activity in vitro. Inducer activity isdefined herein as QR inducing activity in Hepa lclc7 cells (murinehepatoma cells) incubated with extracts of sprouts, seeds or other plantparts untreated with myrosinase. Inducer activity is measured in Hepalclc7 murine hepatoma cells grown in 96-well microtiter plates.Typically 10,000 Hepa lclc7 cells are introduced into each well.Hepatoma cells are grown for 24 hours and a plant extract containingmicrogram quantities of fresh plant tissue is serially diluted acrossthe microtiter plates into fresh culture medium containing 0.15 ml αMEMculture medium amended with 10% Fetal Calf Serum (FCS) and streptomycinand penicillin. The cells are further incubated for 48 hours. QRactivity (based on the formation of the blue-brown reduced tetrazoliumdye) is measured with an optical microtiter plate scanner in celllysates prepared in one plate, and related to its protein concentration.Quantitative information on specific activity of QR is obtained bycomputer analysis of the absorbances. One unit of inducer activity isthe amount that when added to a single microtiter well doubles the QRactivity. (See Prochaska and Santamaria, Anal. Biochem. 169: 328-336(1988) and Prochaska et al., Proc. Natl. Acad. Sci. USA 89: 2394-2398(1992)).

Inducer potential or Phase 2 enzyme-inducing potential is a measure ofthe combined amounts of inducer activity in plant tissue provided byisothiocyanates, plus glucosinolates that can be converted by myrosinaseto isothiocyanates. Glucosinolates are not themselves inducers ofmammalian Phase 2 enzymes, whereas isothiocyanates are inducers. Inducerpotential therefore is defined herein as QR activity in murine lclc7hepatoma cells incubated with myrosinase-treated extracts of thesprouts, seeds or other plant parts. In the present invention thereforeinducer potential is measured by means of the murine hepatoma cellbioassay of QR activity in vitro as described above. Inducer potentialis measured in Hepa lclc7 murine hepatoma cells grown in 96-wellmicrotiter plates. Typically, 10,000 Hepa lclc7 cells are introducedinto each well. Hepatoma cells are grown for 24 hours and a plantextract containing microgram quantities of fresh plant tissue isserially diluted across the microtiter plates into fresh culture mediumcontaining 0.15 ml αMEM culture medium amended with 10% Fetal Calf Serum(FCS) and streptomycin and penicillin. Myrosinase (6 units/ml plantextract) is added to the plant extract. Myrosinase is purified bymodification of the technique of Palmieri et al., Anal. Biochem. 35:320-324 (1982) from 7 day old Daikon sprouts grown on agar supportcontaining no added nutrients. Following 234-fold purification, themyrosinase had a specific activity of 64 units/mg protein unit=amount ofenzyme required to hydrolyze 1 μmol sinigrin/min!. Plant extract isdiluted 200-fold into the initial wells of the microtiter plate followedby 7 serial dilutions. The cells are further incubated for 48 hours. QRactivity (based on the formation of the blue-brown reduced tetrazoliumdye) is measured with an optical microtiter plate scanner in celllysates prepared in one plate, and related to its protein concentration.Quantitative information on specific activity of QR is obtained bycomputer analysis of absorbances. One unit of inducer potential is theamount that when added to a single microtiter well doubles the QRactivity. (See Prochaska and Santamaria, Anal. Biochem. 169: 328-336(1988) and Prochaska et al., Proc. Natl. Acad. Sci. USA 89: 2394-2398(1992)).

A monofunctional inducer increases the activity of Phase 2 enzymesselectively without significantly altering Phase 1 enzyme activities.Monofunctional inducers do not depend on a functional Ah receptor butenhance transcription of Phase 2 enzymes by means of an AntioxidantResponsive Element (ARE).

A cruciferous sprout is a plant or seedling that is at an early stage ofdevelopment following seed germination. Cruciferous seeds are placed inan environment in which they germinate and grow. The cruciferous sproutsof the instant invention are harvested following seed germinationthrough and including the 2-leaf stage. The cruciferous sprouts ofinstant invention have at least 200,000 units per gram fresh weight ofPhase 2 enzyme-inducing potential at 3-days following incubation underconditions in which cruciferous seeds germinate and grow.

II. Description

A major mechanism of protection provided by fruits and vegetables inreducing the cancer incidence in humans depends on minor chemicalcomponents which, when delivered to mammalian cells, elevate levels ofPhase 2 enzymes that detoxify carcinogens. It has now been discoveredthat the anticarcinogenic activity of certain edible plants can beincreased. Plants such as Brassica oleracea variety italica (broccoli)are normally not harvested until they form heads. By growing theseplants only to the seedling or sprout stage, that is between the onsetof germination and the 2-leaf stage, the levels of inducers of enzymesthat detoxify carcinogens and protect against cancer can be increased atleast five-fold over those found in commercial stage vegetables of thesame cultivars. Often increases of between 10 and 1000-fold have beenobserved.

Harvesting plants at an early seedling or sprout stage, or otherwisearresting their growth, leads to the greatest inducer potential andyields a food product of a type to which consumers are alreadyaccustomed. The Phase 2 enzyme-inducing potential of such sprouts may beas much as several hundred times higher than that observed in adult,market stage vegetables obtained from the same seeds. Thus it ispossible that humans can consume the same quantities of inducerpotential by eating relatively small quantities of sprouts, rather thanlarge quantities of market-stage vegetables.

It has now been found that most of the inducer potential of cruciferplants is due to their content of isothiocyanates and their biogenicprecursors, glucosinolates. Glucosinolates are converted toisothiocyanates by the enzyme myrosinase which is a thioglucosidase.Normally myrosinase and glucosinolates are separated in the cell and ifthe cell is damaged, with loss of compartmentalization, myrosinase comesinto contact with glucosinolates, which are then converted toisothiocyanates.

In order to screen large numbers of edible plants and to evaluate theeffects of environmental perturbation on Phase 2 enzyme-inducerpotential in those vegetables, it was necessary to improve upon thepreviously described techniques for homogenization and extraction ofthose vegetables. Techniques initially described for the extraction ofPhase 2 inducers from vegetables involved homogenization of thevegetables in cold water, lyophilization, extraction of the resultantpowder with acetonitrile, filtration and evaporative concentration,Prochaska et al., Proc. Natl. Acad. Sci. USA 89: 2394-2398 (1992).

Following identification of sulforaphane as the principal Phase 2inducer from broccoli, comparative extractions were performed into hot80% methanol, yielding similar inducer activity as the aforementionedacetonitrile extracts. When myrosinase was added to these hot methanolextracts in which glucosinolates are freely soluble, there was adramatic enhancement of the Phase 2 inducer activity of these extracts(data summarized in Table 1). The deliberate conversion of theseglucosinolates to isothiocyanates using exogenous myrosinase thus gave abetter index of the inducers for Phase 2 enzymes of the vegetablestested. It was thus clear that the majority of the potential Phase 2inducers in crucifers was usually present in whole plants as theglucosinolate precursors of isothiocyanates.

The preponderance of glucosinolates and the rapidity with which, uponwounding of cruciferous plant tissue, glucosinolates are converted toisothiocyanates, led to the development of an improved extractionprocedure. By manipulation of solvent mixtures and of the water activityof fresh vegetable/solvent homogenates, a procedure was developed thatpermits both glucosinolate and isothiocyanate quantification from thesame, non-concentrated sample. In addition to being the rate-limitingstep in an extraction protocol, evaporative concentration allowsvolatile inducers to escape detection. The improved procedure is bothsimple and efficient, requiring only that the plant sample be completelyhomogenized in solvent. Using this technique, the present inventors havethus been able to demonstrate dramatic increases in the recovery ofinducer activity and inducer potential from cruciferous vegetables overpreviously described techniques.

If fresh-picked vegetables are promptly and gently harvested, directlyinto organic solvents comprising a mixture of DMF/ACN/DMSO and atemperature that prevents myrosinase activity, both glucosinolates andisothiocyanates are efficiently extracted into the organic solventmixture. Preferably, the DMF, ACN and DMSO are mixed in equal volumes.However, the volumes of the three solvents in the mixture can be variedto optimize extraction of specific glucosinolates and isothiocyanatesfrom any plant tissue. The temperature of the extraction mixture ispreferably less than 0° C., and most preferably less than -50° C. Thetemperature of the extraction solvent must be kept above freezing. Atthe same time the enzyme myrosinase, which invariably accompanies theseconstituents in the plants and rapidly converts glucosinolates intoisothiocyanates, is inactive. Such extracts typically contain highquantities of glucosinolates and negligible quantities ofisothiocyanates. The in planta myrosinase activity varies betweendifferent plant species.

Glucosinolates are not themselves inducers of mammalian Phase 2 enzymes,whereas isothiocyanates are monofunctional inducers in the murinehepatoma cell bioassay of QR activity. The inducer potential, asdistinct from inducer activity, of plant extracts can be measured byadding purified myrosinase, obtained from the same, or other plantsources, to the assay system.

Glucosinolates are converted at least partially to isothiocyanates inhumans. If, however, it is desirable to accelerate this conversion,broccoli or other vegetable sprouts, high in glucosinolates, can bemixed with myrosinase. The mixture can be in water, or some othernon-toxic solvent that does not inactivate myrosinase. The myrosinasecan be from a partially purified or purified preparation. Alternatively,the myrosinase can be present in plant tissue, such as a small quantityof crucifer sprouts rich in myrosinase, including Raphanus sativus ordaikon. Such a preparation can be used to produce a "soup" for ingestionthat is high in isothiocyanates and low in glucosinolates. Inducerpotential can be measured using a multiwell plate screen with murinehepatoma cells for in vitro measurement of QR specific activity asdescribed above.

The ratio of monofunctional to bifunctional inducer activity of planttissue is measured by bioassaying plant extracts, as described above,not only in wild-type Hepa lclc7 cells, but also, in mutants designatedcl and BP^(r) cl that have either defective Ah receptors or defectivecytochrome P₁ -450 genes, respectively. Prochaska and Talalay, CancerResearch 48: 4776-4782 (1988).

A harvested sprout according to the present invention can beincorporated immediately into food products such as fresh salads,sandwiches or drinks. Alternatively, the growth of the harvested sproutcan be arrested by some active human intervention, for example byrefrigeration, at a stage of growth prior to the 2-leaf stage, typicallybetween 1 and 14 days after germination of seeds. Growth arrest can alsobe accomplished by removing a sprout from its substrate and/or watersource. Freezing, drying, baking, cooking, lyophilizing and boiling areamong the many treatments that can be used to arrest growth. These mayalso be useful for either preserving myrosinase activity in the sprout(e.g., lyophilizing) or for inactivating myrosinase activity in thesprout (e.g., boiling), as is desired in a particular application.

The harvested sprout can also be allowed to mature further, underdifferent growing conditions, prior to incorporation into a foodproduct. For example, the sprout can be harvested at a very young age ofdevelopment, such as 1 to 2 days after seed inhibition. The sprout canthen be allowed to mature under different growing conditions, such asincreased or decreased light intensity, temperature or humidity;exposure to ultraviolet light or other stresses; or addition ofexogenous nutrients or plant growth regulators (hormones). The sprout isthen immediately incorporated into a food product, such as for freshconsumption in salads. Alternatively, the growth of the sprout isarrested and/or further treated by means of lyophilization, drying,extracting with water or other solvents, freezing, baking, cooking, orboiling, among others.

A sprout is suitable for human consumption if it does not havenon-edible substrate such as soil attached or clinging to it. Typicallythe sprouts are grown on a non-nutritive solid support, such as agar,paper towel, blotting paper, Vermiculite, Perlite, etc., with water andlight supplied. Thus, if a sprout is not grown in soil, but on a solidsupport, it does not need to be washed to remove non-edible soil. If asprout is grown in a particulate solid support, such as soil,Vermiculite, or Perlite, washing may be required to achieve a sproutsuitable for human consumption.

Sprouts can be grown in containers which are suitable for shipping andmarketing. Typically such containers are plastic boxes or jars whichcontain a wetted pad at the bottom. The containers allow light topenetrate while providing a mechanically protective barrier. Numerousmethods for the cultivation of sprouts are known, as exemplified by U.S.Pat. Nos. 3,733,745, 3,643,376, 3,945,148, 4,130,964, 4,292,760 or4,086,725. Food products containing the sprouts of the instant inventioncan be stored and shipped in diverse types of containers such as jars,bags and boxes, among many others.

Sprouts suitable as sources of cancer chemoprotectants are generallycruciferous sprouts, with the exception of cabbage (Brassica oleraceacapitata), cress (Lepidiumsativum), mustard (Sinapis alba and S. niger)and radish (Raphanus sativus) sprouts. The selected sprouts aretypically from the family Cruciferae, of the tribe Brassiceae, and ofthe subtribe Brassicinae. Preferably the sprouts are Brassica oleraceaselected from the group of varieties consisting of acephala (kale,collards, wild cabbage, curly kale), medullosa (marrowstem kale), ramosa(thousand head kale), alboglabra (Chinese kale), botrytis (cauliflower,sprouting broccoli), costata (Portuguese kale), gemmifera (Brusselssprouts), gongylodes (kohlrabi), italica (broccoli), palmifolia (Jerseykale), sabauda (savoy cabbage), sabellica (collards), and selensia(borecole), among others.

Particularly useful broccoli cultivars to be used in the claimed methodare Saga, DeCicco, Everest, Emerald City, Packman, Corvet, Dandy Early,Emperor, Mariner, Green Comet, Green Valiant, Arcadia, CalabreseCaravel, Chancellor, Citation, Cruiser, Early Purple Sprouting RedArrow, Eureka, Excelsior, Galleon, Ginga, Goliath, Green Duke,Greenbelt, Italian Sprouting, Late Purple Sprouting, Late WinterSprouting White Star, Legend, Leprechaun, Marathon, Mariner, Minaret(Romanesco), Paragon, Patriot, Premium Crop, Rapine (Spring Raab),Rosalind, Salade (Fall Raab), Samurai, Shogun, Sprinter, Sultan, Taiko,and Trixie. However, many other broccoli cultivars are suitable.

Particularly useful cauliflower cultivars are Alverda, Amazing, Andes,Burgundy Queen, Candid Charm, Cashmere, Christmas White, Dominant, Elby,Extra Early Snowball, Fremont, Incline, Milkyway Minuteman, Rushmore,S-207, Serrano, Sierra Nevada, Siria, Snow Crown, Snow Flake, SnowGrace, Snowbred, Solide, Taipan, Violet Queen, White Baron, WhiteBishop, White Contessa, White Corona, White Dove, White Flash, WhiteFox, White Knight, White Light, White Queen, White Rock, White Sails,White Summer, White Top, Yukon. However, many other cauliflowercultivars are suitable.

Suitable sprouts will have at least 200,000 units per gram of freshweight of Phase 2 enzyme-inducing potential following 3-days incubationof seeds under conditions in which the seeds germinate and grow.Preferably the sprouts will have at least 250,000 units of inducerpotential per gram of fresh weight, or even 300,000 units, 350,000units, 400,000 units, or 450,000 units. Some samples have been found tocontain greater than 500,000 units per gram of fresh weight at 3-days ofgrowth from seeds.

The level of inducing activity and inducing potential has been found tovary among crucifers and even among cultivars. Most preferably, thesprouts are substantially free of indole glucosinolates and theirbreakdown products which have Phase 1 enzyme-inducing potential inmammalian cells, and substantially free of toxic levels of goitrogenicnitriles and glucosinolates such as hydroxybutenyl glucosinolates, whichupon hydrolysis yield oxazolidonethiones which are goitrogenic. MatureBrussels sprouts and rapeseed are rich in these undesirableglucosinolates.

Non-toxic solvent extracts according to the invention are useful ashealthful infusions or soups. Non-toxic or easily removable solventsuseful for extraction according to the present invention include water,liquid carbon dioxide or ethanol, among others. The sprouts can beextracted with cold, warm, or preferably hot or boiling water whichdenature or inactivate myrosinase. The residue of the sprouts,post-extraction, may or may not be removed from the extract. Theextraction procedure may be used to inactivate myrosinase present in thesprouts. This may contribute to the stability of the inducer potential.The extract can be ingested directly, or can be further treated. It can,for example, be evaporated to yield a dried extracted product. It can becooled, frozen, or freeze-dried. It can be mixed with a crucifervegetable which contains an active myrosinase enzyme. This willaccomplish a rapid conversion of the glucosinolates to isothiocyanates,prior to ingestion. Suitable vegetables that contain active myrosinaseare of the genus Raphanus, especially daikon, a type of radish.

Seeds, as well as sprouts have been found to be extremely rich ininducer potential. Thus it is within the scope of the invention to usecrucifer seeds in food products. Suitable crucifer seeds may be groundinto a flour or meal for use as a food or drink supplement. The flour ormeal is incorporated into breads, other baked goods, or health drinks orshakes. Alternatively, the seeds may be extracted with a non-toxicsolvent such as water, liquid carbon dioxide or ethanol to preparesoups, teas or other drinks and infusions. The seeds can also beincorporated into a food product without grinding. The seeds can be usedin many different foods such as salads, granolas, breads and other bakedgoods, among others.

Food products of the instant invention may include sprouts, seeds orextracts of sprouts or seeds taken from one or more different crucifergenera, species, varieties, subvarieties or cultivars. It has been foundthat genetically distinct crucifers produce chemically distinct Phase 2enzyme-inducers. Different Phase 2 enzyme-inducers detoxify chemicallydistinct carcinogens at different rates. Accordingly, food productscomposed of genetically distinct crucifer sprouts or seeds, or extractsor preparations made from these sprouts or seeds, will detoxify abroader range of carcinogens.

Glucosinolates and/or isothiocyanates can be purified from seed or plantextracts by methods well known in the art. See Fenwick et al., CRC Crit.Rez. Food Sci. Nutr. 18: 123-201 (1983) and Zhang et al., Pro. NatlAcad. Sci. USA. 89: 2399-2403 (1992). Purified or partially purifiedglucosinolate(s) or isothiocyanate(s) can be added to food products as asupplement. The dose of glucosinolate and/or isothiocyanate added to thefood product preferably is in the range of 1 μmol to 1,000 μmols.However, the dose of glucosinolate and/or isothiocyanate supplementingthe food product can be higher.

The selection of plants having high Phase 2 enzyme-inducer potential insprouts, seeds or other plant parts can be incorporated into Cruciferaebreeding programs. In addition, these same breeding programs can includethe identification and selection of cultivars that produce specificPhase 2 enzyme-inducers, or a particular spectrum of Phase 2enzyme-inducers. Strategies for the crossing, selection and breeding ofnew cultivars of Cruciferae are well known to the skilled artisan inthis field. Brassica Crops and Wild Allies: Biology & Breeding; S.Tsunoda et al. (eds), Japan Scientific Societies Press, Tokyo pp. 354(1980). Progeny plants are screened for Phase 2 inducer activity or thechemical identity of specific Phase 2 enzyme-inducers produced atspecific plant developmental stages. Plants carrying the trait ofinterest are identified and the characteristic intensified or combinedwith other important agronomic characteristics using breeding techniqueswell known in the art of plant breeding.

EXAMPLE 1 Comparison of Cruciferous Strout Inducting Potential

Sprouts were prepared by first surface sterilizing seeds of differentspecies from the cruciferae family with a 1 min treatment in 70%ethanol, followed by 15 min in 1.3% sodium hypochlorite containingapproximately 0.001% Alconox detergent. Seeds were grown in sterileplastic containers at a density of approximately 8 seeds/cm² for from 1to 9 days on a 0.7% agar support that did not contain added nutrients.The environment was carefully controlled with broad spectrum fluorescentlighting, humidity and temperature control. The seeds and sprouts wereincubated under a daily cycle of 16 hours light at 25° C. and 8 hoursdark at 20° C.

Sprouts were harvested following 3-days of incubation and immediatelyplunged into 10 volumes of a mixture of equal volumes of DMF/ACN/DMSO at-50° C. This solvent mixture has a freezing point of approximately -33°C., but when admixed with 10% water, as found in plant material, thefreezing point is depressed to below -64° C. The actual freezing pointdepression is even greater with plant material.

Homogenization was accomplished either by manually grinding the samplesin a glass-on-glass homogenizer in the presence of a small amount of thetotal solvent used, then gradually adding more solvent or homogenizingthe sample in 10 volumes of solvent using a Brinkman PolytronHomogenizer for 1 min at half-maximum power. The homogenate was thencentrifuged to remove remaining particulates and stored at -20° C. untilassayed.

Inducer potential of plant extracts prepared as described above, wasdetermined by the microtiter plate bioassay method as described in theDefinitions section above.

Broccoli and cauliflower sprouts harvested and assayed at 3-days afterincubation of seeds under growth conditions have Phase 2 enzyme-inducerpotential greater than 200,000 units/g fresh weight. On the other hand,cabbage, radish, mustard and cress have Phase 2 enzyme-inducer potentialof less than 200,000 units/g fresh weight when assayed at the same timepoint.

EXAMPLE 2 Variation In Inducer Potential Among Different BroccoliCultivars

There is variation in inducer potential among different broccolicultivars. In addition, most of the inducer potential in crucifers ispresent as precursor glucosinolates. The inducer activity and inducerpotential of market stage broccoli heads was determined followingDMF/ACN/DMSO extractions and assay of QR activity as described above.

Bioassay of homogenates of such market stage broccoli heads, with andwithout the addition of purified plant myrosinase, showed that theamount of QR activity found in the absence of myrosinase was less than5% of that observed with added myrosinase. These observations confirmedprevious suggestions (see Matile et al., Biochem. Physiol. Pflanzen 179:5-12 (1984)) that uninjured plants contain almost no freeisothiocyanates.

                  TABLE 1                                                         ______________________________________                                        Effect of Myrosinase on Inducer Activity                                      of Market-Stage Broccoli Plant Heads                                                          Units per gram (wet weight)                                   Broccoli        vegetable                                                     cultivar        -myrosinase                                                                             +myrosinase                                         ______________________________________                                        DeCicco         5,882     37,037                                              Calabrese Corvet                                                                              1,250     41,666                                              Everest         *          8,333                                              Dandy Early     *         20,000                                              Emperor         *         13,333                                              Saga            5,000     13,333                                              Emerald City    *         12,500                                              ______________________________________                                         *Below limits of detection (833 units/g)                                 

As can be observed in Table 1, most of the plant inducer potential isderived from glucosinolates following hydrolysis by myrosinase to formisothiocyanates. Hence, hydrolysis is required for biological activity.

EXAMPLE 3 Inducer Potential is Highest in Seeds and Decreases as SproutsMature

Phase 2 enzyme-inducer potential is highest in seeds and decreasegradually during early growth of seedlings. Plants were prepared byfirst surface sterilizing seeds of Brassica oleracea variety italicacultivars Saga and DeCicco with a 1 min treatment in 70% ethanol,followed by 15 min in 1.3% sodium hypochlorite containing approximately0.001% Alconox detergent. Seeds were grown in sterile plastic containersat a density of approximately 8 seeds/cm² on a 0.7% agar support thatdid not contain added nutrients. The environment was carefullycontrolled with broad spectrum fluorescent lighting, humidity andtemperature control. The seeds and sprouts were incubated under a dailycycle of 16 hours light at 25° C. and 8 hours dark at 20° C.

Each day plants were rapidly and gently collected from the surface ofthe agar from replicate containers. The plants were harvested gently tominimize glucosinolate hydrolysis by endogenous myrosinase released uponplant wounding. Samples containing approximately 40 sprouts werehomogenized in 10 volumes of DMF/ACN/DMSO solvent at -50° C. whichdissolves nearly all the non-lignocellulosic plant material.

Harvested plants were homogenized and QR activity with and withoutmyrosinase, was determined as described above. As can be seen in FIG. 1,Phase 2 enzyme-inducer potential per gram of plant is highest in seeds,but decreases gradually following germination. No detectable (less than1000 units/g) QR inducer activity was present in the absence of addedmyrosinase.

EXAMPLE 4 Sprouts Have Higher Inducer Potential Than Market Stage Plants

The cruciferous sprouts of the instant invention have higher Phase 2enzyme-inducer potential than market stage plants. More specifically,sprouts have at least a 5-fold greater Phase 2 enzyme-inducing potentialthan mature vegetables. For example, total inducing potential of7-day-old broccoli sprouts, extracted with DMF/ACN/DMSO and treated withmyrosinase, as described above, were 238,000 and 91,000 units/g freshweight, compared to 25,000 and 20,000 units/g fresh weight forfield-grown heads of broccoli cultivars Saga and DeCicco, respectively.

Sprout extracts of over 40 different members of the Cruciferae have nowbeen bioassayed and broccoli sprouts remain the most Phase 2enzyme-inducer-rich plants tested. Total inducing potential of organicsolvent extracts of market stage and sprout stage broccoli and daikon isshown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Comparison of Inducer Potential in                                            Sprouts and Mature Vegetables                                                         Activity (units/g fresh weight)                                       Vegetable Mature                                                              Fold                                                                          Cultivar* Vegetable    Sprout**  Difference                                   ______________________________________                                        DAIKON                                                                        Miura       625         26,316   42                                           Tenshun    3,333        33,333   10                                           Hakkai     1,471        16,667   11                                           Ohkura     2,857        50,000   18                                           BROCCOLI                                                                      Saga      25,000       476,000   19                                           DeCicco   25,000       625,000   25                                           Everest    8,333       1,087,000 130                                          Emerald City                                                                            12,500       833,000   67                                           Packman   20,000       556,000   28                                           ______________________________________                                         *The commercial portion of each plant was sampled (e.g. the taproot of        Raphanus sativus variety radicola  radish!), and heads of Brassicsa           oleracea variety italica  broccoli!. Myrosinase was added to all extracts     tested.                                                                       **Broccoli sprouts were 1day old and daikon seedlings were 4-5days old.  

Sprouts of the broccoli cultivar Everest contained 130-fold more inducerpotential (units/g fresh weight) than mature vegetables. The induceractivity in broccoli was significantly higher than in daikon.

EXAMPLE 5 Inducer Potential of Broccoli Sprout Extracts

Inducer potential of a series of water extracts of 3-day old broccolisprouts of the cultivar Saga were determined. Plants were prepared byfirst surface sterilizing seeds of Brassica oleracea variety italica(broccoli) cultivar Saga by a 1 min treatment in 70% ethanol, followedby 15 min in 1.3% sodium hypochlorite containing approximately 0.001%Alconox detergent. Seeds were grown in sterile plastic containers at adensity of approximately 8 seeds/cm² for 72 hours on a 0.7% agar supportthat did not contain added nutrients. The environment was carefullycontrolled with broad spectrum fluorescent lighting, humidity andtemperature control (16 hours light, 25° C./8 hours dark, 20° C.).

Plants were rapidly and gently collected from the surface of the agar tominimize glucosinolatehydrolysis by endogenous myrosinase released uponplant wounding. Sprouts (approximately 25 mg fresh wt/sprout) weregently harvested and immediately and rapidly plunged into approximately3 volumes of boiling water in order to inactivate endogenous myrosinaseas well as to extract glucosinolates and isothiocyanates from the planttissue. Water was returned to a boil and maintained at a rolling boilfor 3 min. The sprouts were then either strained from the boiledinfusion (tea, soup) or homogenized in it, and the residue then removedby filtration or centrifugation.

Data in Table 3 represent both homogenates and infusions. Preparationswere stored at -20° C. until assayed. Inducer potential of plantextracts, prepared as described above, was determined as described inDefinitions section above.

                  TABLE 3                                                         ______________________________________                                        Inducer Potentials of Hot Water Extracts                                      of 3-Day Saga Broccoli Sprouts                                                EXTRACT NO.   units/g fresh weight                                            ______________________________________                                         1            500,000                                                          2            370,000                                                          3            455,000                                                          4            333,000                                                          5            435,000                                                          6            333,000                                                          7            625,000                                                          8            250,000                                                          9            313,000                                                         10            357,000                                                         11            370,000                                                         12            370,000                                                         13            217,000                                                         14            222,000                                                         15            1,000,000                                                       16            714,000                                                         17            435,000                                                         18            1,250,000                                                       19            263,000                                                         AVERAGE       464,000 ± 61,600 S.E.M.                                      ______________________________________                                    

Some variability in the amount of Phase 2 enzyme-inducer potential wasdetected. High levels of Phase 2 enzyme-inducer potential, however, wereconsistently observed.

EXAMPLE 6 Hot Water Broccoli Extracts Treated With Daikon Myrosinase

QR activity in a hot water broccoli extract increased in the presence ofa vegetable source of myrosinase. An aqueous extraction of 3-day oldsprouts of broccoli cultivar Saga grown on water agar, in whichmyrosinase was inactivated by boiling for 3 min, was divided into 6different 150 ml aliquots. Nine-day old daikon sprouts, a rich source ofthe enzyme myrosinase, were added to this cooled infusion in amountsequivalent to 0, 5, 9, 17, 29 and 40% (w/w) of the broccoli. QRactivity, as determined in the Definition section, of the controlextracts containing 0% daikon was 26,300 units/gram fresh weight whileQR activity of the extracts that had received daikon as a source ofmyrosinase ranged from 500,000 to 833,000 units/gram fresh weight ofbroccoli. Accordingly, myrosinase present in the daikon sprouts,increased the QR activity in the broccoli extract greater than 19-fold.

EXAMPLE 7 Glucoraphanin and Glucoerucin Are The PredominantGlucosinaolates In Hot Water Extracts Of Broccoli (Cultivar Saga)Sprouts

Paired Ion Chromatography (PIC). Centrifuged hot water extracts of3-day-old broccoli (cultivar Saga) sprouts were subjected to analyticaland preparative PIC on a reverse phase C18 Partisil ODS-2 HPLC column inACN/H₂ O (1/1, by vol.) with tetraoctylammonium (TOA) bromide as thecounter-ion. Only three well-separated peaks were detected: peak Aeluted at 5.5 min, B at 11.5 min, and C at 13 min at a molar ratio(A:B:C) of ca. 2.5: 1.6:1.0 (monitored by UV absorption at 235 nm), andthey disappeared if the initial extracts were first treated with highlypurified myrosinase. Peaks A, B, and C contained no significant induceractivity, and cyclocondensation assay of myrosinase hydrolysates showedthat only Peaks A and C produced significant quantities ofisothiocyanates, accounting for all the inducer activity. See Zhang etal., Anal. Biochem. 205: 100-107 (1992). Peak B was not furthercharacterized. Peaks A and C were eluted from HPLC as TOA salts butrequired conversion to ammonium salts for successful mass spectroscopy,NMR and bioassay. The pure peak materials were dried in a vacuumcentrifuge, redissolved in aqueous 20 mM NH₄ Cl, and extracted withchloroform to remove excess TOA bromide. The ammonium salts ofglucosinolates remained in the aqueous phase, which was then evaporated.

Identification of Glucosinolates. The ammonium salts of Peaks A and Cwere characterized by mass spectrometric and NMR techniques: (a)negative ion Fast Atom Bombardment (FAB) on a thioglyerol matrix; thisgave values of 436 (Peak A) and 420 (Peak C) amu for the negativemolecular ions, and (b) high resolution NMR, as shown in FIG. 2,provided unequivocal identification of the structure. Peak A isglucoraphanin (4-methylsulfinylbutyl glucosinolate), and Peak C is theclosely related glucoerucin (4-methythiobutyl glucosinolate). Theseidentifications and purity are also consistent with the inducerpotencies; Peaks A and C, after myrosinase hydrolysis had potencies of36,100 and 4,360 units/μmol, respectively, compared with reported CDvalues of 0.2 μM (33,333 units/μmol) for sulforaphane and 2.3 μM (2,900units/μmol) for erucin. CD values are the concentrations of a compoundrequired to double the QR specific activity in Hepa lclc7 murinehepatoma cells. Since there are no other glucosinolate peaks, and theinducer activity of peak A and C account for the total inducer activityof the extracts, it is therefore likely that in this cultivar ofbroccoli, there are no significant quantities of other inducers, i.e.,no indole or hydroxyalkenyl glucosinolates. Further, the isolatedcompounds are therefore substantially pure.

EXAMPLE 8 Comparison Of Aqueous and Organic Solvent Techniques ForExtraction of Inducer Potential

Plants were prepared by first surface sterilizing seeds of Brassicaoleracea variety italica (broccoli) cultivar Saga, with 70% ethanolfollowed by 1.3% sodium hypochlorite and 0.001% alconox. The seeds weregrown in sterile plastic containers at a density of approximately 8seeds/cm² for 72 hours on a 0.7% agar support that did not contain addednutrients. The environment was carefully controlled with broad spectrumfluorescent lighting, humidity, and temperature control (16 hours light,25° C./8 hours dark, 20° C.).

The plants were rapidly and gently collected from the surface of theagar to minimize glucosinolatehydrolysis by endogenous myrosinasereleased upon plant wounding. A portion of the plants was homogenizedwith 10 volumes of the DMF/ACN/DMSO solvent at -50° C., as described inExample 1, which dissolves nearly all the non-lignocellulosic plantmaterial. Alternatively, the bulk of the harvested plants was plungedinto 5 volumes of boiling water for 3 min to inactivate endogenousmyrosinase and to extract glucosinolates and isothiocyanates. The cooledmixture was homogenized, centrifuged, and the supernant fluid was storedat -20° C.

Inducer potential of plant extracts, prepared by the two methodsdescribed above, was determined by the microtiter plate bioassay asdescribed above. Typical inducer potentials in an average of 5preparations were 702,000 (DMF/ACN/DMSO extracts) and 505,000 (aqueousextracts) units/g fresh weight of sprouts.

Spectrophotometric quantitation of the cyclocondensation product of thereaction of isothiocyanates with 1,2-benzenedithiole was carried out asdescribed in Zhang et al., Anal. Biochem. 205: 100-107 (1992).Glucosinolates were rapidly converted to isothiocyanates after additionof myrosinase. About 6% of the total hot water extractable materialdissolved solids! consisted of glucosinolates. These results demonstratethat (a) isothiocyanate levels in the crude plant extracts are extremelylow; (b) myrosinase rapidly converts abundant glucosinolates toisothiocyanates; (c) hot water extraction releases over 70% of theinducer activity extractable with a triple solvent mixture permittingrecovery of most of the biological activity in a preparation that issafe for human consumption; and (d) over 95% of the inducing potentialin the intact plant is present as glucosinolates and therefore no otherinducers are present in biologically significant quantities.

EXAMPLE 9 Developmental Regulation of Glucosinolate Production

Preliminary experiments in which field grown broccoli (cultivar DeCicco)was harvested at sequential time points from the same field indicatedthat on a fresh weight basis, inducer potential declined from the earlyvegetative stage through commercial harvest, but appeared to increase atlate harvest (onset of flowering). These data suggested that inducerpotential might be highest in seeds. Subsequent studies have shown thatwhen seeds of 8 broccoli cultivars were surface sterilized and grownunder gnotobiotic conditions, Phase 2 enzyme-inducer potential washighest in seeds and declined progressively (on a fresh weight basis)over time throughout the first 14 days of seedling growth.

Expressed on a per plant basis, however, activity remained constant overthis period, suggesting that at this early stage of growth there was nonet synthesis of glucosinolates. However, when the glucosinolateprofiles of market stage broccoli heads and 3 day old sprouts (cultivarEmperor) were compared, there was a profound difference in the apparentglucosinolate compositions of these plants.

Sprouts were prepared by first surface sterilizing seeds of Brassicaoleracea variety italica (broccoli) cultivar Emperor with a 1 minutetreatment in 70% ethanol, followed by 15 min in 1.3% sodium hypochloritewith approximately 0.001% Alconox detergent. Seeds were grown in sterileplastic containers at a density of approximately 8 seeds/cm² for 72hours on a 0.7% agar support that did not contain added nutrients. Theenvironment was carefully controlled; broad spectrum fluorescentlighting, humidity and temperature control (16 hours light, 25° C./8hours dark, 20° C.).

Plants were rapidly and gently collected from the surface of the agar tominimize glucosinolatehydrolysis by endogenous myrosinase released uponplant wounding. Sprouts (approximately 25 mg fresh wt/sprout), weregently harvested and immediately and rapidly plunged into approximately3 volumes of boiling water in order to inactivate endogenous myrosinaseas well as to extract glucosinolates and isothiocyanates from the planttissue. Water was returned to a boil and maintained at a rolling boilfor 3 min. The sprouts were then strained from the boiled infusion (tea,soup) and the infusion was stored at -20° C. until assayed.

Market stage heads were obtained by germinating seeds of the sameseedlot in a greenhouse in potting soil, transplanting to an organicallymanaged field in Garrett County, Md. and harvested at market stage.Heads were immediately frozen upon harvest, transported to thelaboratory on ice and extracts were prepared in an identical fashion tothose described above for sprouts except that approximately 3 gramfloret tissue samples were used for extraction.

Inducer potential of plant extracts, prepared as described above, wasdetermined by the microtiter plate bioassay method as described inExample 1. Paired ion chromatography revealed two major peaks, probablyglucobrassicin and neo-glucobrassicin, in extracts of market stage headswith similar retention times to glucobrassicin (indole-3-ylmethylglucosinolate) and neo-glucobrassicin (1-methoxyindole-3-ylmethylglucosinolate). This observation is consistent with published reports onthe glucosinolate composition of mature broccoli plants. However, pairedion chromatography under the same conditions of identically preparedextracts of 3-day-old sprouts showed absence of glucobrassicin orneo-glucobrassicin. Additionally, 3-day-old sprouts of differentbroccoli cultivars produce different mixtures of glucosinolates.Accordingly, glucosinolate production is developmentally regulated.

EXAMPLE 10 Evaluation of Anticarcinogenic Activities of Broccoli SproutPreparations In the Huggins Dmba (9,10 Dimethyl-1,2-Benzanthracene)Mammary Tumor Model

Sprouts were prepared by first surface sterilizing seeds of Brassicaoleracea variety italica (broccoli) cultivar Saga with a 1 min treatmentin 70% ethanol, followed by 15 min in 1.3% sodium hypochlorite withapproximately 0.001% Alconox detergent. Seeds were grown in sterileplastic containers at a density of approximately 8 seeds/cm² for 72hours on a 0.7% agar support that did not contain added nutrients. Theenvironment was carefully controlled with broad spectrum fluorescentlighting, humidity and temperature control (16 hours light, 25° C./8hours dark, 20° C.).

The plants were rapidly and gently collected from the surface of theagar to minimize glucosinolate hydrolysis by endogenous myrosinasereleased upon plant wounding. A large quantity of sprouts was harvestedby immediately and rapidly plunging into approximately 3 volumes ofboiling water in order to inactivate endogenous myrosinase, as well asextracting glucosinolates and isothiocyanates from the plant tissue.Water was returned to a boil and maintained at a rolling boil for 3 min.Sprouts were then strained from the boiled infusion (tea, soup) and theinfusion was lyophilized and stored as a dry powder at -20° C.(designated Prep A). Other sprouts, similarly prepared were extractedwith boiling water, cooled to 25° C. and were amended with a quantity of7 day old daikon sprouts equivalent to approximately 0.5% of theoriginal fresh weight of broccoli sprouts. This mixture was homogenizedusing a Brinkman Polytron Homogenizer and incubated at 37° C. for 2hours following which it was filtered through a sintered glass filter,lyophilized as above and stored as a dried powder at -20° C. (designatedPrep B).

QR inducer activity and inducer potential of plant extracts, prepared asdescribed above, was determined by the microtiter plate bioassay methodas described above. The induction of QR activity in preparation A islargely due to glucosinolates; predominantly glucoraphanin, which is theglucosinolate of sulforaphane, but this preparation also contains someglucoerucin, which is the sulfide analog of glucoraphanin. The inductionQR activity of preparation B is almost exclusively due toisothiocyanates arising from treatment of glucosinolates withmyrosinase.

Female Sprague-Dawley rats received at 35 days of age were randomized; 4animals per plastic cage. All animals received 10 mg DMBA, by gavage in1 ml sesame oil, at age 50 days. Sprout preparations (A or B) or vehiclecontrol were given by garage at 3, 2 & 1 day prior to DMBA, on the dayof DMBA (2 hr prior to the DMBA dose) and on the day following DMBAdosing. The vehicle used was 50% Emulphor 620P/50% water. Animals weremaintained on a semi-purified AIN-76A diet ad lithium from the time ofreceipt until termination of the experiment (167 days of age).

                  TABLE 4                                                         ______________________________________                                        ANTICARCINOGENIC ACTIVITIES OF BROCCOLI SPROUT                                EXTRACTS IN THE DMBA RAT MAMMARY TUMOR MODEL                                                  NUMBER OF         MULTIPLICITY:                                               ANIMALS AT TOTAL  NUMBER                                            TREAT-    TERMI-     TUMOR  OF TUMORS                                   GROUP MENT      NATION     NUMBER PER RAT                                     ______________________________________                                        CON-  DMBA only 19         34     1.79                                        TROL                                                                          PRE-  324 mg/dose                                                                             18         19     1.05                                        PAR-  (100 μmol                                                            ATION sulforaphane                                                            A     equiv.)                                                                 (Gluco-                                                                       sin-                                                                          olate)                                                                        PRE-  424 mg/dose                                                                             20         11     0.55                                        PAR-  (100 μmol                                                            ATION sulforaphane                                                            B     equiv.)                                                                 (Iso-                                                                         thio-                                                                         cyanate)                                                                      ______________________________________                                    

The development of palpable tumors was delayed for as much as 5 weeks bythe administration of sprout extracts. Rats treated with eitherPreparation A or B had significantly fewer tumors than the untreatedcontrol, and the multiplicity of tumors (tumors per rat) wassignificantly lower in the animals receiving Preparations A or B.

EXAMPLE 11 Metabolism and Clearance Of Glucosinolates in Humans

Two male, non-smoking volunteers ages 35 and 40 years, each in goodhealth, were put on a low vegetable diet in which no green or yellowvegetables, or condiments, mustard, horseradish, tomatoes or papayaswere consumed. After 24 hours on such a diet, all urine was collected in8 hr aliquots. After 24 hours of baseline data, subjects ingested 100 mlof broccoli sprout soup (prepared as below), containing 520 μmol ofglucosinolates.

The sprouts were prepared by first surface sterilizing seeds of Brassicaoleracea variety italica (broccoli) cultivar Saga with a 1 min treatmentin 70% ethanol, followed by 15 min in 1.3% sodium hypochlorite with ca.0.001% Alconox detergent. Seeds were grown in sterile plastic containersat a density of approximately 8 seeds/cm² for 72 hours on a 0.7% agarsupport that did not contain added nutrients. The environment wascarefully controlled with broad spectrum fluorescent lighting, humidityand temperature control (16 hours light, 25° C./8 hours dark, 20° C.).The plants were rapidly and gently collected from the surface of theagar to minimize glucosinolate hydrolysis by endogenous myrosinasereleased upon plant wounding. A large quantity of sprouts was harvestedby immediately and rapidly plunged into approximately 3 volumes ofboiling water in order to inactivate endogenous myrosinase as well as toextract glucosinolates and isothiocyanates from the plant tissue. Waterwas returned to a boil and maintained at a rolling boil for 3 min.Following the boiling step, sprouts were homogenized directly in theirinfusion water for 1 min using a Brinkman Polytron Homogenizer and thepreparations were frozen at -79° C. until use.

Inducer potential of plant extracts, prepared as described above, wasdetermined by the microtiter plate bioassay method as described above.Inducer potential is nearly all due to glucosinolates; predominantlyglucoraphanin, which is the glucosinolate of sulforaphane, but someglucoerucin which is the sulfide analog of glucoraphanin was alsopresent. When converted to isothiocyanates by the addition of purifiedmyrosinase, Phase 2 enzyme-inducing potential was 100,000 units/ml andcontained 5.2 μmol of isothiocyanates per ml, as determined by thecyclocondensation reaction described in Example 7. Thus, the subjectsconsumed a total of 520 μmol of glucosinolates.

Collection of 8 hour urine samples was continued for an additional 30hours. Urinary excretion of isothiocyanate conjugates (dithiocarbamates)was monitored using the cyclocondensation reaction as described inExample 7.

                  TABLE 5                                                         ______________________________________                                        EXCRETION OF DITHIOCARBAMATES BY TWO SUBJECTS                                 INGESTING 520 MICROMOLES OF GLUCOSINOLATES                                    EXTRACTED FROM SAGA BROCCOLI                                                                  SUBJECT 1                                                                             SUBJECT 2                                             TIME    CONDITION   μmol Dithiocarbamate                                   Collection Time per 8 hour urine                                              (hours)         collection                                                    ______________________________________                                         8      baseline      1.4       2.7                                           16      baseline      2.1       0.9                                           24      baseline      1.7       5.4                                           32      1st 8 hour    23.2      20.4                                                  post-dose                                                             40      2nd 8 hour    9.9       36.8                                                  post-dose                                                             48      3rd 8 hour    4.4       14.0                                                  post-dose                                                             56      4th 8 hour    4.2       4.1                                                   post-dose                                                             Total post-dose minus 39.8      63.2                                          average baseline:                                                             Total as Percent of dose:                                                                           6.7%      12.2%                                         ______________________________________                                    

The two subjects studied metabolically converted a significant fractionof the ingested glucosinolates to the isothiocyanates which wereconverted to cognate dithiocarbamates and measured in the urine.

EXAMPLE 12 Effects of Physical Interventions on Sprout Growth onProduction of Inducers of Quinone Reductase

Sprouts were prepared by first surface sterilizing seeds of Raphanussativum (daikon) by a 1 minute treatment with 70% ethanol, followed by a15 min treatment with 1.3% sodium hypochlorite with approximately 0.001%Alconox detergent. Seeds were grown in sterile plastic containers at adensity of approximately 8 seeds/cm² for 7 days on a 0.7% agar supportthat did not contain added nutrients. The environment was carefullycontrolled with broad spectrum fluorescent lighting, humidity andtemperature control (16 hours light 25° C./8 hours dark, 20° C.).

Treated sprouts were irradiated with germicidal UV light for 0.5 hr ondays 5 and 6. Treated sprouts were only half the height of the untreatedcontrols. Plants were harvested on day 7 by rapidly and gentlycollecting the plants from the surface of the agar to minimizeglucosinolate hydrolysis by endogenous myrosinase released upon plantwounding. Sprouts were harvested by immediate and rapid plunging intoapproximately 10 volumes of DMF/ACN/DMSO (1:1:1) at approximately -50°C. in order to inactivate endogenous myrosinase as well as to extractglucosinolates and isothiocyanates. Sprouts were immediately homogenizedwith a ground glass mortar and pestle and stored at -20° C.

Inducer potential of plant extracts, prepared as described above, wasdetermined by the microtiter plate bioassay method as described above.Inducer potential of the UV-treated sprouts was over three times that ofuntreated controls. Treatment of sprouts with ultraviolet lighttherefore increased the Phase 2 enzyme-inducer potential of the planttissue.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims. All publications and patentapplications mentioned in this specification are indicative of the levelof skill of those in the art to which the invention pertains.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference in its entirety.

What is claimed is:
 1. A method of preparing a food product rich inglucosinolates, comprising germinating cruciferous seeds, with theexception of cabbage, cress, mustard and radish seeds, and harvestingsprouts prior to the 2-leaf stage, to form a food product comprising aplurality of sprouts.
 2. The method according to claim 1, wherein saidsprouts contain non-toxic levels of indole glucosinolates and theirbreakdown products and goitrogenic hydroxybutenyl glucose isolates. 3.The method according to claim 1, wherein said seeds are a Brassicaoleracea selected from the group of varieties consisting of acephala,alboglabra, botrytis, costata, gemmifera, gongylodes, italica,medullosa, palmifolia, ramosa, sabauda, sabellica, and selensia.
 4. Themethod according to claim 3, wherein said seeds are Brassica oleraceavariety italica.
 5. The method according to claim 3, wherein said seedsare Brassica oleracea variety botrytis.
 6. The method according to claim5, wherein said seeds are Brassica oleracea variety botrytis subvarietycauliflora.
 7. A method of preparing a food product, comprisingextracting glucosinolates and isothiocyanates from cruciferous sprouts,with the exception of cabbage, cress, mustard and radish sprouts,harvested prior to the 2-leaf stage, with a non-toxic solvent, removingthe extracted sprouts from said solvent, and recovering the extractedglucosinolates and isothiocyanates.
 8. A method of preparing a foodproduct according to claim 7, wherein active myrosinase enzyme is mixedwith said cruciferous sprouts, or said extracted glucosinolates andisothiocyanates, or both said cruciferous sprouts or said extract.
 9. Amethod of preparing a food product rich in glucosinolates, comprisinggerminating cruciferous seeds that produce sprouts having at least200,000 units per gram fresh weight of Phase 2 enzyme-inducing potentialwhen measured after 3-days of growth and which contain non-toxic levelsof indole glucosinolates and their breakdown products and goitrogenichydroxybutenyl glucosinolates, and harvesting sprouts prior to the2-leaf stage to form a food product comprising a plurality of sprouts.10. The method according to claim 9, wherein said seeds are a Brassicaoleracea selected from the group of varieties consisting of acephala,alboglabra, botrytis, costata, gemmifera, gongylodes, italica,medullosa, palmifolia, ramosa, sabauda, sabellica, and selensia.
 11. Themethod according to claim 10, wherein said seeds are Brassica oleraceavariety italica.
 12. The method according to claim 10, wherein saidseeds are Brassica oleracea variety botrytis.
 13. The method accordingto claim 12, wherein said seeds are Brassica oleracea variety botrytissubvariety cauliflora.
 14. A method of preparing a food product,comprising introducing cruciferous seeds, wherein said seeds producesprouts having at least 200,000 units per gram fresh weight of Phase 2enzyme-inducing potential when measured after 3-days of growth andnon-toxic levels of indole glucosinolates and their breakdown productsand goitrogenic hydroxybutenyl glucosinolates, into another edibleingredient.
 15. A method of preparing a food product comprisingextracting glucosinolates and isothiocyanates with a non-toxic solventand isothiocyanantes from cruciferous seeds, sprouts, plants or plantparts wherein seeds that produce said sprouts, plant, or plant parts,have at least 200,000 units per gram fresh weight of Phase 2enzyme-inducing potential when measured after 3-days of growth andwherein said seeds, sprouts, plants or plant parts have non-toxic levelsof indole glucosinolates and their breakdown products and goitrogenichydroxybutenyl glucosinolates, and recovering the extractedglucosinolates and isothiocyanates.
 16. A method of preparing a foodproduct according to claim 15, wherein active myrosinase enzyme is mixedwith said cruciferous seeds, sprouts or plants; or said extractedglucosinolates and isothiocyanates; or both said cruciferous seeds,sprouts or plants and said extract.